Transposable elements are ubiquitous in higher eukaryotic genomes. Approximately 45% of the human genome are covered by transposable elements (Lander, E. S. et al. (2001) Nature, 409, 860-921). Transposable elements are DNA sequences that can be mobilized and spread to different positions within the genome of a single cell, a process called transposition. In the process, they can cause mutations and change the amount of DNA in the genome. Transposable elements are mobile genetic elements which are also called “jumping genes”. There is a variety of mobile genetic elements, and they can be grouped based on their mechanism of mobilization. Class I mobile genetic elements, or retrotransposons, are transcribed into RNA, and then reverse transcribed and reintegrated into the genome, thereby duplicating the element (“copy & paste” mechanism). The major classes of retrotransposons either contain long terminal repeats at both ends (LTR retrotransposons) or lack LTRs and possess a polyadenylate sequence at their 3′ termini (non-LTR retrotransposons). Class II mobile genetic elements are termed DNA transposons and are generally excised from one genomic site and integrated into another by a “cut & paste” mechanism.
The majority of transposable elements in mammalian genomes are retrotransposons, which are considered to transpose via an RNA intermediate. Of these, the largest group comprises non-LTR retrotransposons which cover ˜42% of the human genome (Lander et al. 2001, Nature 409, 880ff). Long interspersed elements (LINES) are a major class of non-LTR retrotransposons and cover approximately 21% of the genome. LINE-1 retrotransposons (L1) cover about 17% of the human genome (Lander et al., 2001, Nature 409, 860-921) and play a significant role in shaping the mammalian genome, not only through their own expansion but also through the mobilization of non-L1 sequences. While the average haploid human genome harbors ˜516,000 L1 copies, the subgroup of active L1s is fairly small, encompassing 80-100 elements (Sassaman et al., 1997, Nat. Genet. 16: 37-43; Brouha et al., 2003, PNAS 100: 5280-5285). So far, 82 retrotransposition-competent, full-length L1 elements were isolated and characterized (Sassaman et al., 1997, Nat. Genet. 16: 37-43; Brouha et al., 2003, PNAS 100: 5280-5285, Moran et al. 1996, Cell 87: 917-927; Kimberland et al. 1999, Hum. Mol. Genet. 8:1557-1560; Brouha et al. 2002; Am. J. Hum. Genet. 71: 327-336). L1s affected the genome by (i) insertion of truncated L1s into new sites, (ii) intrachromosomal homologous recombination between L1s, (iii) transduction of 3′-flanking sequences during retrotransposition, (iv) aiding trans generation of processed pseudogenes and retrotransposition of Alu elements, and (v) by causing genome instability through substantial deletions (Gilbert et al. 2002, Cell 110: 315-325; Symer et al. 2002, Cell 110: 327-338; Babushok & Kazazian, 2007, Human Mutation 28: 527-539).
A retrotransposition-competent, functional L1 element (RC-L1, FIG. 1) covers ˜6.1 kb and contains a 5′ untranslated region (5′ UTR) with an internal and endogenous, CpG-rich promoter, a 1 kb ORF1 encoding a protein (p40) of ˜40 kD with RNA-binding capability, followed by a 3.8 kb ORF2 coding for a protein (p150) with a predicted molecular weight of ca 150 kD with endonuclease (EN) and reverse transcriptase (RT) activities and a cysteine-histidine-rich domain. The 3′-end of L1 is terminated by a short 3′ UTR, and a poly(A) tail (Ostertag & Kazazian, 2001, Annu. Rev, Genet. 35:501-538) (FIG. 1A). L1 mRNAs are atypical of mammalian RNAs because they are bicistronic and the mechanism of translation of L1 is not understood. The two ORFs are in frame and separated by a 63-bp noncoding spacer region. Mutational analyses demonstrated that both ORF1- and ORF2-encoded functional proteins are required for retrotransposition (Moran et al. 1996, Cell 87: 917-927; Feng et al. 1996, Cell 87: 905-916). At least three functions of the ORF2-encoded protein were shown to be essential for retrotransposition, RT activity, EN activity and a function associated with the cysteine-histidine-rich motif. Insertion of a new L1 copy into the loose genomic target sequence 5′-TTTT/A-3′ (Gilbert et al. 2002, Cell 110:315-325; Feng et al. 1996, Cell 87: 905-916; Jurka, 1997, PNAS 94: 1872-1877; Cost & Boeke, 1998, Biochemistry 37:18081-18093) is initiated by a process termed target-primed reverse transcription (Luan et al. 1993, Cell, 72: 595-605; Cost et al. 2002, EMBO J. 21: 5899-5910). The structure of the target site duplications (TSDs) flanking de novo L1 integrants suggests a model for second strand synthesis of L1 termed “microhomology-driven single strand annealing” (Symer et al. 2002, Cell 110: 327-3389; Martin & Bushman, 2001, Mol. Cell. Biol. 21: 467-475).
While the majority of L1 and other L1-mediated insertions land in intergenic and intronic sequences with little or no consequence for their host, occasional insertions have disrupted gene expression and caused genetic disorders and cancer (for review Babushok & Kazazian, 2007; Human Mutation 28:527-539; Ostertag & Kazazian, 2006. Retrotransposition and Human Disorders. In: Encyclopedia of Life Sciences: John Wiley & Sons. Ltd: Chichester http://www.els.net/[doi: 10.1038/npg.els.0005492]. Of 53 known disease-causing insertions, 17 were caused by L1 itself, while L1-mediated integrations of Alu and SVA elements caused another 33 cases; three additional cases were caused by L1-mediated insertions of simple polyA repeats. For example, germ line L1 insertions into the factor VIII and dystrophin gene gave rise to hemophilia A and muscular dystrophy, respectively (Kazazian et al., 1988, Nature 332:164-166; Narita et al., 1993, J. Clinical Invest. 91:1862-1867; Holmes et al., 1994, Nature Genetics 7:143-148). 16 somatic L1-mediated retrotransposition events caused a variety of cancers, including ALL1 rearrangement leukemias and BRCA1-associated familial breast cancer (Deininger & Batzer, 1999, Mol. Genet. Metab. 67: 183-193). Somatic L1 insertions into the c-myc and APC tumor suppressor gene were shown to be involved in breast and colon cancer, respectively (Morse et al., Nature 333:87-90; Miki et al., 1992, Cancer Research 52:643-645). Thus, L1 is a potential mutagen and L1 retrotransposition is mutagenic.
However, the controlled application of LINE-1 as a tool for random mutagenesis was hampered by the lack of an inducible system which allows temporally defined, quantitative and reversible regulation of high level LINE-1 retrotransposition in mammalian cells.
So far, only constitutive retrotransposition of marked LINE-1 reporter cassettes was achieved in mammalian cell lines and in germ cells or entire organism of transgenic animals (An et al., 2006, Proc. Natl. Acad. Sci., 103: 18662-7; Ostertag, 2002, Nat. Genet. 32: 655-60; Prak et al., 2003, Proc. Natl. Acad. Sci., 100: 1832-7; Babushok et al., 2006, Genome Ress., 16: 240-50). Also, LINE-1 mediated gene transfer was only performed with vectors expressing L1 constitutively (Kubo et al., 2006, Proc. Nad. Acad. Sci., 103: 8036-41; Soifer et al., 2001, Hum. Gene Ther., 12: 1417-28).
WO 88/03169 is merely concerned with constructs useful in yeast and describes a method for inducing retotransposition of yeast Ty retrotransposons in yeast using the inducible GAL1 promoter which is not functional in mammalian cells. Ty elements are LTR retrotansposons that are functional in yeast whereas LINE-1s are non-LTR transposons which are functional in mammalian cells. Consequently, the disclosed techniques can not be transferred to mammalian cells.
Other methods relating to the constitutive expression of LINE-1s are known in the prior art. For example, US 2003/0121063 relates to a method for generating a mutation in the offspring of an animal. To achieve this goal, mice were transfected with different non-inducible LINE1 constructs and the offspring of these transgenic animals was analysed.
In addition, US 2006/0183226 discloses a new method the target-specific introduction of certain LINE-like retrotransposons (TRAS and SMART family members) into mammals. The use of inducible promoters is not described. The patent application US 2006/0183226 covers a similar field and describes several genetic modifications of LINE-1 to achieve sequence specific targeting with the modified construct.
The objective problem is to provide a vector construct and method which allows inducible, tightly controlled and conditional expression of functional tagged non-LTR retrotransposons, in particular, LINE-1 retrotransposons in mammals. The solution of this problem is the provision of a nucleic acid with a tetracycline-response element operably linked to a promoter which in turn is operably linked to a tagged LINE-1 element.